PROJECT SUMMARY TGF-? family proteins are critically important for human health. They regulate differentiation, proliferation and homeostasis in normal cell physiology, but mutations or abnormal expression are associated with devastating diseases, including cancers, and Pulmonary Arterial Hypertension (PAH). Discovering therapeutics that target TGF-? family proteins is an urgent priority and will have a great impact on human health. However, identifying therapeutics has been difficult, because the physiological and pathological activities of TGF-? family proteins are poorly defined. A profound molecular understanding of TGF-? family action is essential to overcome these obstacles. Thus, the overarching goal of the lab is to determine how TGF-? family signaling is regulated and to link our findings with physiological and pathological processes. We blend tools of structural biology, biochemistry and cell biology. Here, we have two aims. In Aim 1 we will validate the newly established mechanism of ligand recognition by Cripto-1 family co-receptors and examine the roles of membrane-anchored and soluble Cripto-1 in cancers. Cripto-1 is a membrane-anchored, EGF-CFC family `co-receptor' that promotes tumor progression. It can both potentiate and inhibit TGF-? family signaling. But how Cripto-1 mediates these opposing activities is not clear. Our recent studies suggest a mechanism. Cripto-1 blocks binding of TGF-? family ligands to receptors. Thus, a soluble form inhibited signaling. But membrane-anchored Cripto-1 potentiated signaling and promoted tumorigenesis. We hypothesize that membrane-anchored Cripto-1 captures ligands at the cell surface and directs ligands into endosomes for signal potentiation and oncogenic activation. Elucidating the Cripto-1/EGF-CFC mechanism of ligand capture and oncogenic activation could reveal epitopes and modalities to target Cripto-1 in cancers. In Aim 2 we will identify the ligand binding determinants of the `type II' TGF-? family receptor BMPRII and establish functional consequences of ligand binding-competition for BMPRII mediated signaling and PAH. TGF-? family signals are transduced via SMAD transcription factors. In PAH, BMPRII mediated SMAD1/5/8 signaling is reduced. Just how is not understood. Our recent findings offer a clue. We discovered that ligands regulated by Cripto-1 or its homolog Cryptic bind BMPRII with high affinity. Until now, BMPRII was believed to be a low affinity receptor for BMPs, a subclass of TGF-? family ligands. We also found that high affinity ligands inhibit BMP dependent SMAD1/5/8 signaling by competing for receptor binding. We hypothesize that Cripto-1/Cryptic ligands suppress BMPRII mediated SMAD1/5/8 signaling in PAH by competing with BMPs for BMPRII binding. Demonstrating that binding- competition promotes PAH phenotypes and identifying epitopes on ligands that contact BMPRII could lead to development of inhibitors that prevent high affinity BMPRII ligands from competing with low affinity BMPs, and thus could help restore BMPRII mediated SMAD1/5/8 signaling in PAH. Successful completion of these studies could help open new avenues for targeting Cripto-1 in cancers and for restoring BMPRII function in PAH.